Analysis of DNA binding proteins associated with hemin-induced transcriptional inhibition. The hemin response element binding protein is a heterogeneous complex that includes the Ku protein.
Hemin inhibits transcription of the tartrate resistant acid phosphatase (TRAP) gene. Using deletion mutagenesis of the mouse TRAP 5'-flanking region, we previously identified a 27-bp DNA segment containing a central GAGGC tandem repeat sequence (the hemin response element [HRE]), which bound nuclear proteins (hemin response element binding proteins [HREBPs]) from hemin-treated cells and appeared to be responsible for mediating transcriptional inhibition in response to hemin. We now have used affinity binding to HRE-derivatized beads to identify four HREBP components with apparent molecular masses of 133-, 90-, 80-, and 37-kD, respectively. The 80- and 90-kD components correspond to the p70 and p80/86 subunits of Ku antigen (KuAg) as documented by partial amino acid microsequencing of tryptic digests and immunologic reactivity. Based on reactivity of the HREBP gel shift band with antibodies to the redox factor protein (ref1) in shift Western experiments, it is shown that the 37-kD component represents ref1. The 133-kD component appeared to be a unique protein. KuAg participation in HREBP complexes was specific as it was present in HREBPs bound to HRE microcircles. Results of depletion/reconstitution experiments suggested that KuAg does not bind alone or directly to HRE DNA, but does so only in conjunction with the 133- and/or 37-kD proteins. We conclude that HREBP is a heterogeneous complex composed of KuAg, ref1, and a unique 133-kD protein. We speculate that the role of heme may be to promote interactions among these components, thereby facilitating HRE binding and downregulation of hemin responsive genes.
Reddy
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Division of Hematology, Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78284-7880, USA.
Alcantara
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D000135:Acid Phosphatase / Q000235:genetics; D034961:Antigens, Nuclear; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D006427:Hemin / Q000494:pharmacology*; D006801:Humans; D000072200:Ku Autoantigen; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000032:analysis* / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D013644:Tartrates / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects*; D014466:Ultraviolet Rays
D034961:Antigens, Nuclear; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D013644:Tartrates; D006427:Hemin; D004247:DNA; D000135:Acid Phosphatase; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; C029768:tartaric acid